Over its eleven-year run, Mystery Science Theater 3000 saw the arrival and departure of various characters. It also featured many recurring guest characters. Below is a listing of both the main characters and the recurring characters from the series.

Jack Perkins (Michael J. Nelson) - in real life the host of the A&E Network's Biography program, Perkins first appeared in MST3K simply to annoy the Mads by describing the movie with glowing praise. When MST3K appeared in syndication as The Mystery Science Hour, Nelson's fake "Jack Perkins" hosted the show.

Torgo (Michael J. Nelson) - a monster/henchman (supposed to be a satyr) in Manos: The Hands of Fate, Torgo was among the most frequently returning "guest characters" of MST3K. He got his knees fixed and returned as "Torgo the White" (an obvious parody of Gandalf the White) to accompany TV's Frank to "Second Banana Heaven" and was never seen again (episode 624, "Samson vs. the Vampire Women").

Jan-in-the-Pan (Mary Jo Pehl) - a woman's severed head from the movie The Brain That Wouldn't Die.

Pitch (Paul Chaplin) - a devil from the Mexican movie Santa Claus, Pitch was one of the few characters from the Comedy Central seasons to return in the Sci Fi Channel seasons.

The Nanites (voiced variously by Kevin Murphy, Paul Chaplin, Mary Jo Pehl, and Bridget Jones) - self-replicating, bio-engineered organisms that work on the ship, they are microscopic creatures that reside in the S.O.L.'s computer systems. (They are similar to the creatures in Star Trek: The Next Generation episode "Evolution", which featured "nanites" taking over the Enterprise.) The Nanites made their first appearance in season 8. Based on the concept of nanotechnology, their comical deus ex machina activities included such diverse tasks as instant repair and construction, hairstyling, performing a Nanite variation of a flea circus, conducting a microscopic war, and even destroying the Observers' planet after a dangerously vague request from Mike to "take care of [a] little problem". They also ran a microbrewery. The Nanites were largely forgotten about during the show's last season, and we are not given an explanation of their fate following the series finale.

"Krankor" (Bill Corbett), who appeared in a host segment during the "Prince of Space" episode, and returned three episodes later in a host segment for "Invasion of the Neptune Men", featuring a movie with a similar plot. He was a vain, would-be conqueror with an unfortunately chicken-like appearance and a drawn-out, braying laugh (described by BBI as "like a Buick not turning over").

Synthia (Rebecca Hanson) is a imperfect clone of Pearl Forrester that serves as one of Kinga's henchpersons in Moon 13.

Ardy (Joel Hodgson) aids Kinga in sending the movie to Jonah.

The Skeleton Crew is Kinga's house band that plays in intros and outgos segments. Har Mar Superstar leads the Skeleton Crew as their director.